Field of Invention
The present invention relates to methods and compositions, for reducing the virucidal activity of compositions that would normally exhibit some degree of virucidal activity. By using the methods of the present invention, the virucidal activity of such compositions can be reduced in comparison to the virucidal activity of a composition that does not include the steps of the present invention. More specifically, the present invention relates to methods for producing antigenic Porcine Circovirus Type II (PCV-2) compositions such that they show relatively little or no virucidal activity as compared to the compositions known in the art using current detection methods, and in particular, as compared to compositions not produced by a method according the present invention. The present invention further relates to a novel immunogenic composition, preferably a PCV-2 containing composition produced according to the method provided by the present patent application, preferably characterized by reduced or no virucidal activity relative to comparable compositions described in the art. According to a further aspect, the present invention also provides immunogenic compositions comprising purified PCV-2 antigen, preferably purified PCV-2 antigen with an improved immunogenicity.
Description of the Prior Art
Porcine circovirus type 2 (PCV-2) is a small (17-22 nm in diameter), icosahedral, non-enveloped DNA virus, which contains a single-stranded circular genome. PCV-2 shares approximately 80% sequence identity with porcine circovirus type 1 (PCV-1). However, in contrast with PCV-1, which is generally non-virulent, swine infected with PCV-2 exhibit a syndrome commonly referred to as Post-weaning Multisystemic Wasting Syndrome (PMWS). PMWS is clinically characterized by wasting, paleness of the skin, unthriftiness, respiratory distress, diarrhea, icterus, and jaundice. In some affected swine, a combination of all symptoms will be apparent while other swine will only have one or two of these symptoms. During necropsy, microscopic and macroscopic lesions also appear on multiple tissues and organs, with lymphoid organs being the most common site for lesions. A strong correlation has been observed between the amount of PCV-2 nucleic acid or antigen and the severity of microscopic lymphoid lesions. Mortality rates for swine infected with PCV-2 can approach 80%. In addition to PMWS, PCV-2 has been associated with several other infections including pseudorabies, porcine reproductive and respiratory syndrome (PRRS), Glasser's disease, streptococcal meningitis, salmonellosis, postweaning colibacillosis, dietetic hepatosis, and suppurative bronchopneumonia.
Several vaccine are available to reduced the impact of PCV-2 infections in pigs. U.S. Pat. No. 6,703,023 provides a DNA based vaccine for the prophylaxis of pigs against PMWS. In WO 03/049703 production of a live chimeric vaccine is described, comprising the non-pathogenic PCV1 virus in which, however, the ORF2 protein is replaced by the ORF2 protein of the pathogenic PCV-2. WO 99/18214 and WO 99/29717 have provided several PCV-2 strains and procedures for the preparation of a killed PVC2 vaccine. Preparation of subunit vaccines have also been described in WO 99/18214 and WO 99/29717. An effective ORF2 based subunit vaccine has been reported in WO 06/072065. A further ORF-2 based subunit vaccine is described also in WO 07/28823. However, none of the vaccine described in the prior art includes a non-virucidal and/or purified PCV-2 antigen, preferably a highly purified PCV-2 ORF2 antigen.
Immunogenic compositions against PCV-2 and various immunogenic compositions against other pathogens often have a virucidal effect on other antigens. Current regulatory standards (9 CFR 113.35) permit some virucidal activity in multivalent compositions, but this virucidal activity cannot result in a loss of more than 0.7 logs/ml of a live virus or less than 0.7 logs/ml CFU of live bacteria when combined with the other components of the immunogenic composition. Compositions that have more virucidal activity than permitted cannot be combined with other antigens to create a multivalent vaccine.
Open reading frame 2 (ORF2) protein of PCV-2, having an approximate molecular weight of 30 kDa when run on SDS-PAGE gel, has been utilized in the past as an antigenic component in vaccines and immunogenic compositions for PCV-2. Typical methods of obtaining ORF2 for use in such vaccines and compositions generally consist of amplifying the PCV-2 DNA coding for ORF2, expressing the ORF2 protein within a host cell, and extracting the ORF2 protein from the host cell via cell lysis. The recovered ORF2 cell lysate is then used as the antigenic portion of an immunogenic composition or vaccine. In some cases the ORF2 containing cell lysate is separated from the cell debris.
What is needed is a method for reducing the virucidal activity of PCV-2-containing immunogenic compositions and antigens therein such that regulatory requirements can be met and efficacious multivalent compositions can be administered. What is further needed are methods for decreasing or reducing the virucidal activity and effect of PCV-2-containing compositions on Porcine Reproductive and Respiratory Syndrome Virus (PRRSV). What is still further needed are immunogenic compositions that have undergone the methods of the present invention such that their virucidal activity has been reduced to acceptable standards and can be combined with other antigens to form multivalent immunogenic compositions.